Cerebral malaria (CM) pathogenesis is due to a complex interaction between the vascular, coagulation and immune systems, and parasite sequestration. About 30% of cerebral malaria (CM) patients die despite adequate anti-parasite therapy, indicating that the host response contributes to pathogenesis and that these patients can be rescued by adjunctive therapy aimed at ameliorating the host response. The fine molecular details required for the development of such adjunctive therapies, however, remain poorly defined. By using the experimental cerebral malaria (eCM) model, we have observed that both citrulline and arginine administration protects all recipients from eCM mortality. The levels of amino acids in the blood during eCM indicate a profound disruption of the urea cycle, suggesting that metabolic encephalopathy (ME) with hyperammonemia is likely present. Hyperammonemia has also been reported in P. falciparum patients, which in turn may contribute to cerebral edema that MRI findings indicate is a hallmark of CM in African children. ME likely functions in concert with P. falciparum-specific processes to elicit CM and mortality because P. vivax patients also exhibit some degree of ME. We therefore propose a limited exploratory grant to determine (i) whether urea cycle dysregulation in blood and brain are indeed contributing to eCM pathogenesis, and (ii) whether eCM-protective citrulline and arginine ameliorate the urea cycle dysregulation. Due to exploratory nature of this R21 proposal and the limited budget, we will use a single in vivo model of CM (namely, Plasmodium berghei ANKA infection of mice). If a link is established in eCM, then the rationale and preliminary data for an RO1 project will have been established to confirm these findings are relevant to humans, other mechanisms, such as arginine effects on the immune system, are not of greater importance, and the verification in expensive clinical trials of CM patients. Our preliminary data indicate tha citrulline protects against eCM pathogenesis in part by restoring the urea cycle homeostasis, and citrulline is a substrate in the urea cycle. We hypothesize that citrulline and arginine ameliorate the dysregulation of substrates in the urea cycle caused by infection with Plasmodium and thereby prevents ME; ME leads, in part, to the breakdown of the BBB, vascular leak, and ultimately CM. To address this hypothesis, we propose in aim 1 to determine the extent to which the urea cycle is impaired in eCM, and whether this impairment contributes to eCM pathogenesis. We will also assess hallmarks of ME, namely astrocyte swelling, development of Alzheimer type II astrocytes, elevated glutamine and low glutamate levels, elevated ammonia levels, and oxidative/nitrosative stress in the brain and then correlate these hallmarks with eCM pathogenesis. In aim 2, we will ameliorate eCM hypoargininemia by citrulline or arginine administration that protects all recipients from the genesis of eCM and partially restores the blood arginine levels. We will determine whether this amelioration of blood arginine levels partially restores the urea cycle, reduces the toxic levels of ammonia in the brain and hence protects against the development of eCM. These studies are significant because they directly address pathogenic mechanisms of an important but often overlooked disease that kills millions of people and defines the mechanisms to target for a potential adjunctive therapy to rescue those presenting with cerebral malaria from their disease. These studies are pre- translational in that they define new mechanisms in CM pathogenesis that are amenable to adjunctive therapies and define biomarkers to validate the efficacy of potential adjunctive therapy. Even though not required for a R21 grant, we have extensive preliminary data to support our hypothesis, established all techniques in our laboratory, and have the infrastructure necessary for performance of the proposed studies. My malaria group colleagues at LIDI all enthusiastically support my work and transition to independence, which this limited R21 grant will facilitate.